Abstract

Drug resistance of pathogens is an increasing problem whose underlying mechanisms are not fully understood. Cellular uptake of the major drugs against Trypanosoma brucei spp., the causative agents of sleeping sickness, is thought to occur through an unusual, so far unidentified adenosine transporter. Saccharomyces cerevisiaewas used in a functional screen to clone a gene (TbAT1) from Trypanosoma brucei brucei that encodes a nucleoside transporter. When expressed in yeast,TbAT1 enabled adenosine uptake and conferred susceptibility to melaminophenyl arsenicals. Drug-resistant trypanosomes harbor a defective TbAT1 variant. The molecular identification of the entry route of trypanocides opens the way to approaches for diagnosis and treatment of drug-resistant sleeping sickness.

Reduced drug uptake has emerged as a common characteristic of drug-resistant trypanosomes [(1); reviewed in (2)], rendering the molecular identification of drug transport systems crucial for the understanding of the underlying resistance mechanisms. The main clinical trypanocides are the melaminophenyl arsenical melarsoprol and diamidines. As cellular uptake of these agents has been suggested to occur through a transport system specific for adenosine and adenine [(3–5); reviewed in (6)], we decided to functionally clone the trypanosomal gene or genes encoding an adenosine transporter or transporters. We took advantage of the fact that the yeastSaccharomyces cerevisiaedo not take up exogenous adenosine and cannot use it as a purine source (Fig. 1, A and B). Yeast cells defective in purine biogenesis (ade2) (7) were transformed with a Trypanosoma brucei brucei bloodstream form cDNA expression library (8) and selected for growth in media containing adenosine as sole purine source. Library plasmids conferring the ability to proliferate were isolated (9) and found to encode a putative transporter, designated TbAT1. When expressed in yeast, TbAT1 enabled growth on adenosine as sole purine source (Fig. 1A) and cellular uptake of adenosine (Fig. 1B). Adenosine transport was saturable (Fig. 1C) and conformed to Michaelis-Menten kinetics with an apparent Michaelis constant (Km) of 2.2 μM (10).

Trypanosoma brucei brucei salvage adenosine from their mammalian hosts through two high-affinity transport activities, P1 and P2, that differ in substrate specificity. P1 is specific for adenosine and inosine, whereas P2 transports adenosine, adenine, melaminophenyl arsenicals, and diamidines (3, 4). To determine whether TbAT1 encodes the P1 or P2 activity, we assessed substrate specificity by the ability of potential substrates to inhibit TbAT1-mediated adenosine transport in yeast (11). Adenine caused a strong reduction, whereas inosine, hypoxanthine, guanosine, guanine, uridine, and uracil had no effect (Fig. 2A). Moreover, radioactively labeled inosine was not taken up, and neither inosine nor guanosine could support growth of ade2 yeast expressing TbAT1(12). Among the trypanocides tested for inhibition of adenosine transport, the melaminophenyl arsenicals (melarsoprol and melarsen oxide) and isometamidium (a phenanthridine used in veterinary medicine) were most effective (Fig. 2B), suggesting that these drugs are TbAT1 substrates. The experimental compound tubercidin (7-deazaadenosine) produced a smaller but substantial reduction. The diamidines (pentamidine and diminazene aceturate) had no substantial effect. Thus, the apparent substrate specificity of TbAT1 closely matches that of the reported P2 transport activity (3–5), except for the insensitivity to diamidines. TbAT1 expressed in yeast may lack a trypanosomal cofactor or modification required for diamidine recognition. Future studies of TbAT1 function in genetically engineered trypanosomes will elucidate the role of this transporter in diamidine uptake.

Specificity of TbAT1-mediated transport in yeast. (A) Adenosine and adenine but not other physiological nucleosides and bases compete for adenosine transport. [3H]Adenosine (1 μM) transport was measured in yeast expressing TbAT1, in the presence of the indicated compounds (Ar, adenosine; A, adenine; I, inosine; H, hypoxanthine; Gr, guanosine; G, guanine; Ur, uridine; U, uracil; all at 100 μM). [3H]Adenosine transport in the presence of the potential substrates is presented as the percentage of transport in the absence of competitive substrate. (B) Melaminophenyl arsenicals and isometamidium inhibit adenosine transport. [3H]Adenosine (1 μM) uptake was measured in yeast expressing TbAT1, in the presence of the indicated drugs (MelB, melarsoprol; MelOx, melarsen oxide; Pent, pentamidine; Dim, diminazene aceturate; Iso, isometamidium; Tub, tubercidin; Sin, sinefungin; at 100 μM except for melarsoprol, which was added at 72 μM). (C)TbAT1 expression in yeast renders the cells sensitive to melarsen oxide. Cells carrying TbAT1,TbAT1r, or vector only (control) were grown in minimal media containing 1 mM hypoxanthine, either in the presence of 100 μM melarsen oxide (open bars) or without drug (solid bars). Cell growth was quantified as number of generations during 5 hours of incubation time.

As labeled melaminophenyl arsenicals are unavailable, we could not directly measure transport of these drugs by TbAT1. Instead, we determined whether TbAT1 could mediate uptake of melarsen oxide into cells, measured as susceptibility to the drug (13). Expression of TbAT1 in yeast rendered cell growth sensitive to melarsen oxide (Fig. 2C), suggesting that TbAT1 indeed transports this drug. As expected for competing substrates, the presence of adenosine or adenine in the media abrogated TbAT1-mediated melarsen toxicity (12).

Sequencing of the cDNA revealed a protein of 463 amino acids (Fig. 3A) with a predicted structure of 10 transmembrane α-helices, cytosolic NH3- and CO2-termini, and a large, negatively charged cytosolic loop between transmembrane domains 6 and 7 (Fig. 3B). Recently, two nucleoside transporter genes (LdNT1.1 andLdNT1.2) have been cloned from the protozoan parasiteLeishmania donovani(14).LdNT1.1 and LdNT1.2 are 99.5% identical and tandemly linked (14, 15). In contrast,TbAT1 appears to be a single-copy gene, as determined by Southern (DNA) blot analysis (12). The LdNT1 transporters belong to the ENT (equilibrative nucleoside transporter) family, feature 11 predicted transmembrane domains, and accept a broad range of substrates including pyrimidine nucleosides (16). LdNT1 and TbAT1 are of similar length and share 30% identical amino acid residues (15). Thus, TbAT1 may also be a member of the ENT family, albeit a distant one that shows restricted substrate specificity and, possibly, a distinct membrane topology.

Sequence and predicted structure of TbAT1 and TbAT1r. (A) Deduced amino acid sequence; predicted transmembrane domains are underlined, and point mutations in TbAT1r are indicated in bold above the TbAT1 sequence (22). (B) Predicted TbAT1 topography based on hydropathy and distribution of charges. Potential transmembrane domains are numbered. Hydrophobic residues (Val, Leu, Ile, Phe, and Trp) are represented in black and polar residues (Ser, Thr, Asn, and Gln) in white; positive (Lys, His, and Arg) and negative (Asp and Glu) charges are indicated by symbols. The point mutations in TbAT1r are indicated. (C) Distinction between TbAT1 alleles by restriction digest. PCR with the primers sfa-s and sfa-as amplifies 677 bp of the purine transporter gene. A diagnostic digest with Sfa NI produces fragment sizes of 566 and 111 bp in the case of TbAT1 (from STIB 777S; lane 1, undigested; lane 2, digested) and 435 and 242 bp in the case of TbAT1r (from STIB 777R; lane 3, undigested; lane 4; digested). STIB 777S and STIB 777R are homozygous for the respective mutations. The same digestion pattern as STIB 777R was exhibited by a T. brucei gambiense isolate from an Angolan patient refractory to melarsoprol treatment (lane 5, undigested; lane 6, digested). S, Sfa NI site present only inTbAT1 (encoding Ala178); R, Sfa NI site present only in TbAT1r (encoding Ser286).

Loss or alteration of the P2-type adenosine transporter has been proposed as a mechanism of resistance to melaminophenyl arsenicals (3) and diamidines (4, 5) in trypanosomes. To investigate whether TbAT1 is involved in drug resistance, we cloned and sequenced the genes from T. brucei brucei STIB 777S, a drug-sensitive clone (17), and STIB 777R, a melarsenoxide cysteamine–resistant clone derived from STIB 777S by subcurative treatment in mice (18). Particular care was taken to avoid the artificial introduction of mutations during cloning of the respective genes (19). TbAT1 from STIB 777S was identical in sequence to the originally cloned gene. Sequencing of the TbAT1 allele from STIB 777R (TbAT1r) revealed 10 nucleotide differences, six of which manifest at the amino acid level (Fig. 3, A and B), resulting in the changes Leu71 → Val (L71V) and Leu380→ Pro (L380P) (transmembrane), Ala178 → Thr (A178T) and Gly181 → Glu (G181E) (extracellular), and Asp239 → Gly (D239G) and Asn286 → Ser (N286S) (cytosolic). Introduction of TbAT1r into yeast did not enable usage or uptake of exogenous adenosine (Fig. 1, A and B) and did not confer sensitivity to melarsen oxide (Fig. 2C), suggesting that TbAT1r cannot import adenosine and melaminophenyl arsenicals. In addition, yeast carryingTbAT1r could not grow on other nucleosides as purine source (guanosine, inosine, xanthosine, and purine ribofuranoside) and did not take up radioactively labeled adenine, hypoxanthine, or inosine (12), suggesting that TbAT1r may be a nonfunctional rather than an altered substrate-specificity variant. Not having an antibody against TbAT1, we cannot determine yet whether the amino acid changes in TbAT1r affect transport activity or proper expression of the protein.

Early diagnosis of drug-resistant strains with a simple, sensitive assay would be of great benefit for successful chemotherapy.TbAT1 and TbAT1r are distinguishable by digestion with the restriction endonuclease Sfa NI. While the mutation causing A178T abrogates a Sfa NI site, the mutation underlying N286S creates one (Fig. 3C). Amplification of a fragment of the purine transporter gene by polymerase chain reaction (PCR) followed by Sfa NI digestion (20) may serve as a convenient means for rapid identification of TbAT1r-type drug-resistant trypanosomes. A T. brucei gambiense isolate from a patient refractory to melarsoprol treatment exhibited the Sfa NI digestion pattern typical of TbAT1r (Fig. 3C), suggesting that TbAT1r-like alleles are present in the field. The presence of Thr178 and Ser286was confirmed by direct sequencing of the gene from the T. brucei gambiense field isolate. Sequencing also showed that the T. brucei gambiense allele differs from both TbAT1 andTbAT1r by the absence of the trinucleotide repeat encoding Phe316. This sequence difference rules out the possibility of a contamination with DNA from STIB 777R. Larger studies will be necessary to determine the prevalence of specificTbAT1 alleles and their correlation with melarsoprol treatment failures.

Our findings support the idea that TbAT1 encodes an adenosine transporter mediating uptake of, and thus susceptibility to, melaminophenyl arsenicals and that defects in TbAT1 contribute to resistance to these agents in T. brucei spp. The cloning ofTbAT1 opens prospects for the therapy of sleeping sickness. Current chemotherapy is unsatisfactory because of both the severe side effects of the currently used trypanocides and the considerable fraction of patients that fail to respond to treatment. Functional expression of TbAT1 in yeast provides a tool for the development of drug derivatives with an increased tropism to the parasite (21) and possibly a larger therapeutic window. Identification and characterization of the remaining trypanosomal purine transporters (candidate genes homologous to TbAT1 are present among trypanosome expressed sequence tags) will enable the rational design of drugs and drug combinations that either circumvent drug resistance by using different entry routes or compromise the ability of the parasite to salvage purines.

Out of 106 transformants plated on synthetic minimal media lacking uracil, adenine, and methionine but containing 150 μM adenosine, five colonies grew. Library plasmids were isolated from the five isolates and found to harbor identical inserts: TbAT1 starting at the Eco RI at position +25 from the first ATG (Fig. 3A), a truncated but functional form (12). The 5′ part of the TbAT1 cDNA was obtained by reverse transcriptase PCR with total RNA of T. brucei brucei bloodstream forms, a primer complementary to the TbAT1 coding sequence (GGGCGTAAGGTTCTTCCTTA), and a primer corresponding to the T. brucei brucei spliced leader (CGCTATTATTAGAACAGTTTCTGTAC).

Sensitivity to melarsen oxide was assessed at 30°C in liquid minimal media lacking uracil and methionine, with 1 mM hypoxanthine as sole purine source. Hypoxanthine saturated the yeast endogenous purine permease without affecting TbAT1 function. Melarsen oxide (100 μM) or carrier (dimethyl sulfoxide) alone was added to triplicate cultures. The high concentration of melarsen oxide required for the toxic effect is not surprising given the differences in physiology between S. cerevisiae and T. brucei. The action of melaminophenyl arsenicals is thought to be mediated by conjugation with trypanothione, a biochemical peculiarity of trypanosomatids [

TbAT1 and TbAT1r were amplified by PCR (55°C annealing, 30 cycles) with the primers ant-s (GCCCGGATC CGCTATTATTAGAACAGTTTCTGTAC) and ant-as (GCCCCTCGAGCCGCATGGAGTAAGTCTGA) from genomic DNA of T. brucei brucei STIB 777S and STIB 777R, respectively. For each strain, the products of three independent PCR reactions were pooled, and the pooled DNA was sequenced directly, that is, before cloning. This procedure excluded PCR-introduced sequence errors because any such errors would have had to occur independently in more than one of the three PCR reactions and within the first round of DNA amplification to be detectable by direct sequencing. PCR products were cloned into the yeast expression vector p416-MET25 [

] by means of the incorporated Bam HI (ant1-s) and Xho I (ant1-as) sites. After transformation and testing of activity, plasmids were recovered from yeast and resequenced to confirm that the observed activity (or lack of activity) was attributed to the expected sequence.

A 677–base pair (bp) fragment of the purine transporter gene containing the sites of interest was amplified from genomic DNA by PCR (64°C annealing, 30 cycles) with the primers sfa-s (CGCCGCACTCATCGCCCCGTTT) and sfa-as (CCACCGCGGTGAGACGTGTA). The products of three independent PCR reactions were pooled. Two hundred nanograms of DNA was digested with Sfa NI (New England Biolabs) and analyzed on a 2% agarose gel. The results were reproduced at least three times for each strain, with different DNA preparations and in two different laboratories.

We thank E. Matovu, M. Fasler, and C. Schmid for technical assistance; F. Dietrich for support with sequencing; H. Riezman for the provision of yeast strains; J. Keiser and C. Burri for provision of a melarsoprol-refractory T. brucei gambiense isolate from Angola; V. Eckert and R. Schwarz for the gift of the T. brucei cDNA library; and G. Schatz and U. Müller for comments on the manuscript. This work received financial support from the Stanley Thomas Johnson Foundation. P.M. received fellowships from the Emilia Guggenheim-Schnurr and the Roche Research Foundations; A.K. received funding from the Max Cloëtta Stiftung and the Swiss National Science Foundation. DNA and amino acid sequences of TbAT1 and TbAT1r have been deposited in GenBank under accession numbers and , respectively.